In bacteria, sigma factors (σ) are accessory protein subunits that associate with the RNA polymerase (RNAP) to permit transcription. In Mycobacterium tuberculosis ( Mtb ), the housekeeping σ A -RNAP holoenzyme drives transcription of most genes. Unlike other σs, Mtb σ A contains a ∼200 amino acid N-terminal intrinsically disordered region (IDR) with high charge segregation. Measuring full-length transcription kinetics reveals that removal of the IDR severely reduces steady-state rates of transcription under multi-round conditions, but not under single-round conditions, suggesting that the IDR promotes productive RNAP holoenzyme formation and recycling. To better understand the functional role of the IDR in the context of RNAP interactions and to quantify IDR conformations and dynamics, we turn to fluorescence correlation spectroscopy (FCS) and single-molecule Förster resonance energy transfer (FRET). FCS shows that the IDR markedly enhances RNAP binding. Single-molecule FRET demonstrates that the IDR is compact at low salt and expands with increasing ionic strength (as expected for a strongly charged segregated sequence). The Mtb σ A IDR also undergoes expansion upon RNAP binding, but re-compacts when the σ A -RNAP-DNA complex becomes transcriptionally competent. Nanosecond FCS reveals that the IDR remains highly dynamic in both free and bound states. Global concentration analyses indicate that σ A -RNAP-DNA complex formation directly competes with non-specific σ A -DNA interactions, particularly under conditions of excess DNA or σ A . Finally, as the sequence features of the Mtb σ A IDR are conserved across Actinobacteria, our findings suggest a general mechanism by which dynamic disordered regions modulate transcription in bacteria.
Jensen et al. (Sun,) studied this question.